Isolation of the CAMP gene inducing component.

<p>(<b>A</b>) Heat treatment (100°C) of the hydrophilic fraction of breast milk for 30 min did not affect <i>CAMP</i> gene inducing capacity in HT-29 cells after 48 h of stimulation. Breast milk components were separated into high and low molecular weight components (more or less than 10 kDa) and the <i>CAMP</i> gene inducing capacity of breast milk was retained only in the low molecular weight fraction (≤10 kDa). Displays the mean and SD of at least three independent experiments in triplicate. (<b>B</b>) The low molecular fraction was subjected to cationic exchange and the chromatographic fractions were used for stimulation of HT-29 cells for 48 h. Material in fraction 29, eluted at 4% buffer B, resulted in a 10-fold induction of <i>CAMP</i> gene transcript. (<b>C</b>) Material in fraction 29, from (<b>B</b>), was separated by size exclusion chromatography and obtained fractions were assayed for <i>CAMP</i> gene transcript in HT-29 cells after 48 h of stimulation. A 6.5-fold induction of <i>CAMP</i> gene was observed by stimulation with material from fraction 42. (<b>B–C</b>) The X-axes denote the elution volume. The grey bars representing the activity are shown as a mean of the fold induction performed in triplicate.</p

Relative inductions of CAMP gene transcript by colostrum, transitional milk, mature milk and infant formulas.

<p>(<b>A</b>) HT-29 cells were stimulated for 48 h with 50 g/l hydrophilic fractions of breast milk from different lactation periods. The median of the relative induction of <i>CAMP</i> gene transcript was 2.5-fold for colostrum (1–3 days postpartum, pp), 2.1-fold for transitional milk (4–10 days pp) and 3.9-fold for mature milk (11- days pp) samples. The median of 50 g/l hydrophilic fraction of infant formulas was 2.2, but with a high variability in <i>CAMP</i> gene induction. (<b>B</b>) HT-29 cells were stimulated with the hydrophilic fraction of breast milk collected from one mother from day 7 to 19 pp. A positive linear correlation was observed between <i>CAMP</i> gene transcript induction and time pp (R² = 0.5728, p-value 0.043). (<b>A and B</b>) Each sample is performed in triplicates.</p

The hydrophilic fraction of breast milk induces the CAMP gene in HT-29 cells.

<p>(<b>A</b>) HT-29 cells were stimulated with 5 or 50 g/l hydrophilic fraction of breast milk for 4, 24 or 48 h. After 48 h a 10.8-fold increase of <i>CAMP</i> gene transcript was observed with 50 g/l. (<b>B</b>) Stimulation of HT-29 cells with the hydrophobic fraction of breast milk did not significantly induce <i>CAMP</i> gene expression. (<b>A–B</b>) Show means and standard deviations (SD) of at least three independent experiments performed in triplicates. (<b>C</b>) Western blot analysis showed induction of <i>CAMP</i> gene expression at the protein level. A low constitutive expression of the LL-37 proform hCAP-18 (17 kDa) was observed in the supernatants of unstimulated cells. hCAP-18 concentration increased after stimulation with both 5 and 50 g/l of the hydrophilic fraction with the highest increase after 48 h with 50 g/l. Control (ctrl) was 50 g/l of hydrophilic fraction mixed with cell medium from 48 hours unstimulated cells. Band intensities were calculated from one representative Western blot analysis using Image J and normalized to the band intensity of supernatants from cells propagated for 4 h in medium (US 4).</p

Characterization of the CAMP gene inducing component.

<p>(<b>A</b>) The intact molecular mass of the most abundant components in fraction 42 was 365.1 and 707.2 corresponding to a sodiated monomer of a disaccharide [342.1+ Na]⁺ and a sodiated dimer of a disaccharide [2×342.1+ Na]⁺, respectively. The mass of 343.1 corresponded to a protonated disaccharide [342.1+H]⁺. (<b>B</b>) Fragmentation of the 707.2 component resulted in a peak of 365.1, supporting the presence of disaccharides <i>i.e.</i> [342+Na]⁺. (<b>C</b>) Fragmentation of the 343.1 peak resulted in two peaks of 325.1 and 163.1, corresponding to a disaccharide and a monosaccharide with the elimination of water, respectively. (<b>D</b>) Top: ¹H-NMR spectrum in D₂O (at 20°C) of the pooled fractions 40–42. Bottom: ¹H-NMR spectrum in D₂O (at 20°C), of the equilibrated α/β mixture of lactose. These results establish lactose as the major component in the active fractions.</p

Induction of CAMP gene transcript in different cell lines stimulated with lactose.

<p>(<b>A</b>) HT-29 cells were stimulated with 10–70 g/l lactose for 48 h and <i>CAMP</i> gene transcript was monitored. At 20 g/l a 3.6-fold expression was observed that increased dose-dependently up to 11.3-fold at 70 g/l. (<b>B</b>) HT-29 cells were stimulated with 60 g/l lactose for 4, 24 and 48 h. After 24 h and 48 h an 8- and 17-fold induction of <i>CAMP</i> gene transcript was observed. No induction of <i>CAMP</i> gene was observed at 4 h. (<b>C</b>) T84 cells were stimulated with 60 g/l lactose for 4, 24 and 48 h. A 3.5-fold enhanced level of <i>CAMP</i> gene transcript was observed after 24 h and was 11.3-fold after 48 h. (<b>D</b>) THP-1 monocytes (black line) and differentiated macrophage-like THP-1 cells (grey line) were stimulated with 60 g/l lactose for 4, 24 and 48 h. In monocytes, 7.2 and 25.7-fold induction of <i>CAMP</i> gene transcript was detected after 24 h and 48 h, respectively. The macrophage-like cells exhibited a 13.5-fold induction of <i>CAMP</i> gene transcript after 24 h that declined to 5.6-fold after 48 h. (<b>A–D</b>) Displays the mean and SD of five independent experiments in duplicate.</p

Galactose and lactose levels in feces collected from neonates 1 or 4 weeks after birth. Displays the mean of samples analyzed in triplicate<b>.</b>

<p>Galactose and lactose levels in feces collected from neonates 1 or 4 weeks after birth. Displays the mean of samples analyzed in triplicate<b>.</b></p

CA in spider silk glands.

<p>CA activity and Azure blue staining of histological sections from (A) the sac of a <i>Tegenaria sp</i>. major ampullate gland, (B) <i>E. australis</i> minor ampullate gland, (C) <i>A. diadematus</i> aggregate gland duct, (D) <i>Tegenaria sp</i>. tubuliform gland, and (E) the third limb of the duct of an <i>A. diadematus</i> major ampullate gland. Black precipitates represent CA activity (arrow heads). In (A) the glandular lumen is labeled and the dotted arrow points towards the duct. Nuclei are indicated by (N) and the lumen by (Lu). Scale bar, (A) 50 µm and (B–E) 20 µm.</p

NT and CT respond differently to lowered pH.

<p>Stability of NT and CT (from <i>A. ventricosus</i>) in (A) 20 mM HEPES/MES buffer with 154 mM NaCl and (B) the same buffer without NaCl, measured with Trp fluorescence and CD spectroscopy at 222 nm, respectively, presented as urea concentrations for apparent half-denaturation ([den]^50%, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001921#s3" target="_blank">Materials and Methods</a> for details on how [den]^50% was determined) as a function of pH. The pH region in which CA activity is found in major ampullate glands is indicated by a shaded area in (A).</p

CD spectra of NT and CT.

<p>The residual molar ellipticity was measured at 25, 45, 65, 85, and 95°C and at 25°C after cooling for (A) NT and (B) CT at pH 7.5, 6.5, and 5.5 from top to bottom.</p

pH, bicarbonate, and carbon dioxide in major ampullate glands.

<p>Photograph of a major ampullate gland in which measured pH and HCO₃⁻ values and calculated pCO2 values at different locations are indicated. See Table 1 for details of ISM measurements. Scale bar, 1 mm.</p